Minimizing myocardial damage resulting from ischemic heart disease remains a major challenge for health professionals. Adenosine-mediated cardioprotection has recently been harnessed through a transgenic mouse model of cardiac specific overexpression of the adenosine A1 receptor that demonstrates remarkable protection against ischernia-reperfusion injury. However, the mechanisms of this cardioprotection remain largely unknown. The broad, long-term scientific objective of the proposed research is to use an isolated cardiac myocyte model of adenosine A1 receptor overexpression to investigate the mechanisms of cardioprotection associated with adenosine A1 receptor overexpression. The specific aims of the proposed research are: Specific Aim 1: To characterize the cardioprotection against simulated ischemia associated with adenosine A1 receptor overexpression in H9c2 cells. Using standard transfection techniques to induce adenosine A1 receptor overexpression, H9c2 cells (a rat embryonic heart cell line) will be used to test the hypothesis that adenosine A1 receptor overexpression protects against simulated ischemia. Specific Aim 2: To determine the role of protein kinase C (PKC), tyrosine kinase, p38 mitogen-activated protein kinase (MAPK), and mitochondrial KATP channels in the protection associated with adenosine A1 receptor overexpression in H9c2 cells. PKC, tyrosine kinase, p38 MAPK, and mitochondrial KATP channels contribute to adenosme-mediated cardioprotection; however, their involvement in the protection afforded by adenosine A1 receptor overexpression is largely unknown. These pathways will be inhibited pharmacologically to determine their role in the protection afforded by adenosine A1 receptor overexpression. Specific Aim 3: To determine the effect of adenosine A1 receptor overexpression on the timing of PKC translocation, p38 MAPK phosphorylation, and depolarization of the mitochondrial membrane potential during simulated ischemia. Fluorescence microscopy will be used to observe the effect of adenosine A1 receptor overexpression on the subcellular location of PKC, the dynamics of p38 MAPK phosphorylation, and the time course of mitochondrial depolarization during simulated ischemia. The proposed research, which will introduce undergraduates to the important interface between molecular biology, cardiac physiology, and clinical application, will allow more informed decisions regarding the potential use of adenosine receptor manipulation in humans via gene therapy.